Pseudomonic acid A, also known as mupirocin, is an antibiotic having the formula (I): ##STR1##
It is known that Pseudomonas fluorescens strains are able to biosynthesize, in addition to pseudomonic acid A, other related antibiotics designated by the letters B-D in small quantities [E. B. Chain, G. Mellows, J. Chem. Soc. Perkin Trans I. 318 (1977); J. P. Clayton et al., Tetrahedron Lett., 21, 881 (1980); P. J. O. Hanlon, N. H. Rogers, J. Chem. Soc. Perkin Trans I. 2665 (1983)], represented by formulas (II)-(IV), respectively: ##STR2##
Among the pseudomonic acid antibiotics, from a therapeutic point of view the most valuable is pseudomonic acid A, which has a growth inhibiting effect mainly against Gram positive bacteria (e.g. Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, Klebsiella pneumoniae) and some Gram negative bacteria (e.g., Haemophilus influenzae, Neisseria gonorrhoeae) and its minimal inhibiting concentration is in the range of 0.02-0.5 mg/dm.sup.3. Pseudomonic acid A, by inhibiting the isoleucine-tRNA synthase enzyme, has an effect on the peptide synthesis of pathogen bacteria [J. Hughes and G. Mellows, Biochem. J. 191, 209-219, (1980)]. An advantageous feature of this antibiotic is that it is less toxic for both humans and animals and it is negative in the Ames test. Pseudomonic acid A is presently used in human therapy, in various formulations, for the treatment of skin infections (e.g. impetigo, pyoderma), nose and external ear infections, acne, burns, eczema, psoriasis, in case of ulceration for treatment of secondary infections, and for prevention of hospital infections.
One method for the isolation of pseudomonic acid A from the antibiotic complex-containing culture broth is the liquid-liquid extraction. According to German Patent No. 2,227,739 and U.S. Pat. No. 4,289,703, soluble barium salts are added to the fermentation broth, then the microorganism cells with the insoluble inactive agents are separated by centrifugation and finally the antibiotics are extracted by methyl isobutyl ketone. The antibiotics are then removed from the methyl isobutyl ketone extract by alkaline water and the resulting alkaline aqueous extract is cleaned by reextraction with methyl isobutyl ketone. The crude product obtained is chromatographed, and an ester derivative is prepared from the pseudomonic acid antibiotic complex and purified with preparative thin layer chromatography. The acid form of the pure antibiotic is obtained by hydrolysis.
Belgian Patent No. 870,855 relates to a process in which the culture broth is extracted with methyl isobutyl ketone and from the extract the active substance is extracted by sodium hydrogen carbonate solution. Materials insoluble in alkaline water are separated by filtration, then the pH of the filtrate is acidified and extracted by methyl isobutyl ketone. Finally the pseudomonic acid A is obtained by the concentration of the extract and crystallization from a methyl isobutyl ketone-n-heptane mixture.
Japanese Patent No. 52-70083 relates to two methods for the recovery of pseudomonic acid A from a culture broth. According to one of these methods, the bacterium cells are separated from the culture broth by centrifugation, then the active substance is extracted from the supernatant by ethyl acetate. Then the pseudomonic acid complex is reextracted from the ethyl acetate phase by sodium hydrogen carbonate solution and after acidification it is extracted again with ethyl acetate. After evaporation the residue is purified on silicagel column by the application of chloroform-methanol eluents and finally the pure pseudomonic acid A is reached by crystallization from diisopropyl ether. In the other process the crude product obtained by the above method is chromatographed on DEAE-Sephadex anion exchange column by the application of methanol-ammonia eluent and the pseudomonic acid A containing fractions are separated.
A. D. Curzons described the recovery of pseudomonic acid A from culture broth by lithium salt formation (European Patent No. 0 005 614). The active ingredient-containing methyl isobutyl ketone extract obtained at pH 4.5 is reacted with lithium 2-ethyl-hexanoate dissolved in methanol. Precipitated pseudomonic acid A lithium salt is separated, dissolved in water and the pseudomonic acid A released at pH 4.5 is extracted in methyl isobutyl ketone and precipitated in the presence of n-heptane.
In the methods discussed above, polar and water-immiscible solvents (methyl isobutyl ketone, ethyl acetate, n-butanol) are used for the recovery of the pseudomonic acid complex from the culture broth. According to our experience, selective extraction can not be realized satisfactorily using these processes, since besides the pseudomonic acid complex other polar and nonpolar impurities are also extracted in large quantities. Pseudomonic acid A can be recovered in pure form only with low yield (17-34%) by alkaline extraction of the organic solvents containing the impurities in large quantities, then by organic solvent reextraction at acidic pH, and then by crystallization of the crude product. The use of chromatographic processes for purification is not advantageous on a production scale because of their high labor cost and solvent demand. In addition, in the presence of chromatographic adsorbents and during the chromatographic process intramolecular transformations take place in the active substance, which lead to the formation of the biologically inactive bicyclic compound 9-{4-[1S,6R-8R(1S,3S-dihydroxy-2S-methyl-butyl)-5S-hydroxy-3,7-dioxabicycl o[4.3.0]nonane-4S-yl]-3-methyl-but-2(E)-enoyloxy}nonanoic acid (formula V) ##STR3##
and the compound 9-{4-[1R,6S-4S,10S-dihydroxy-3R-(2S-hydroxy-1S-methyl-propyl)-2,8-dioxabic yclo[4.4.0]decane-9S-yl]-3-methylbut-2(E)-enoyloxy}nonanoic acid (formula VI). ##STR4##
Formation of these compounds and their elimination via recrystallization considerably reduces the recovery yield of the pseudomonic acid A.
Although the isolation process through the intermediate of pseudomonic acid A lithium salt does not contain any chromatographic step, it is not convenient for a production scale process, since the lithium salt used in the process makes the process complicated and expensive to use on a production scale.
Thus there remains a need in the art for a new process for the isolation of the antibiotic pseudomonic acid A, which is free from the disadvantages of the known processes and the application of which in production scale results in a high yield of the recovery of the above-mentioned antibiotic.